Neurochemical, neuroautonomic and neuropharmacological acute effects of sibutramine in healthy subjects

Neurochemical, neuroautonomic and neuropharmacological

acute effects of sibutramine in healthy subjects

Fuad Lechin a,b,c,d,*, Bertha van der Dijs b,c,d, Gerardo Hernandez a,c,d,Beatriz Orozco c,d, Simon Rodriguez c,d, Scarlet Baez b,c

aDepartment of Neurophysiology, Instituto de Medicina Experimental, Faculty of Medicine, Universidad Central de Venezuela,

Caracas, Apartado 80.983, Caracas 1080-A, VenezuelabDepartment of Neurochemistry,, Instituto de Medicina Experimental, Faculty of Medicine,

Universidad Central de Venezuela, Caracas, VenezuelacDepartment of Neuropharmacology, Instituto de Medicina Experimental, Faculty of Medicine,

Universidad Central de Venezuela, Caracas, VenezueladDepartment of Neuroimmunology, Instituto de Medicina Experimental, Faculty of Medicine,

Universidad Central de Venezuela, Caracas, Venezuela

Received 20 July 2005; accepted 27 September 2005

Available online 2 November 2005

Abstract

Sibutramine is a neuropharmacological drug that exerts central (CNS) and peripheral effects including noradrenaline (NA), and serotonin (5-

HT) uptake inhibition. In addition, the drug is able to induce release from DA axons. We measured levels of circulating neurotransmitters in 20

healthy subjects during supine-resting (fasting) state before and after 15 mg of oral sibutramine. Systolic blood pressure (SBP), diastolic blood

pressure (DBP), and heart rate (HR) were also monitored. Sibutramine triggered sustained and progressive increase of NA, NA/Ad ratio and DBP.

Slight increases of DAwere also registered between the 60 and 240 min periods. The rise in DA tended to fade progressively, reaching basal level at

360 min period. Diastolic blood pressure, but neither SBP nor HR, showed significant increases that correlated positively with NA/Ad ratios. Slight

but significant negative correlation was also found between DBP and DA. This correlation tended to fade throughout the trial to show no

significance at the 360 min period. Although neither plasma serotonin (f-5HT) nor platelet serotonin (p-5HT) values showed significant variation

throughout the trial, the f-5HT/p-5HT ratio showed significant decrease throughout. Significant negative correlation was found between f-5HT/p-

5HT ratio and NA/Ad ratio. Our results indicate that sibutramine stimulates neural sympathetic activity but not adrenal sympathetic activity in

healthy individuals. Further, sibutramine lowers parasympathetic activity. The moderate rise in diastolic blood pressure triggered by sibutramine

would be associated with CNS-NA enhancement plus parasympathetic inhibition.

# 2005 Elsevier Inc. All rights reserved.

Keywords: Sibutramine; Plasma noradrenaline; Plasma adrenaline; Plasma dopamine; Blood serotonin; Diastolic blood pressure; Neural sympathetic activity

NeuroToxicology 27 (2006) 184–191

1. Introduction

Sibutramine is a neuropharmacological drug used as an

anorexigenic agent and as such it is widely used to treat obesity,

insulin resistance and polycystic ovary syndrome (PCOS)

(Abolfathi et al., 2004; de Simone et al., 2005; Gaciong and

Placha, 2005; Jordan et al., 2005; Karabacak et al., 2004;

McMahon et al., 2000). Although it is considered a noradrena-

line (NA) + serotonin (5HT) re-uptake inhibitor (Fukagawa and

* Corresponding author. Tel.: + 58 212 961 1048; fax: +58 212 961 0172.

E-mail address: [email protected] (F. Lechin).

0161-813X/$ – see front matter # 2005 Elsevier Inc. All rights reserved.

doi:10.1016/j.neuro.2005.09.004

Sakata, 2001; Invernizi and Garattini, 2004), the drug also

induces release from DA and 5HT axons (Ukai et al., 2004).

Because sibutramine is closely related to amphetamines, it is

viewed as a potentially harmful drug able to trigger adverse

cardiovascular and neuropsychiatric disorders. The fact that its

prolonged administration has provoked enhancement of central

DA activity in rodents has been cited as proof that the drug

behaves as a DA re-uptake inhibitor (Nakagawa et al., 2001).

Although the half-life for sibutramine, M1 and M2

metabolites ranged between 8 and 18 h under fasting conditions,

as in our protocol, it has been demonstrated that feeding is able to

increase the half-life of the drug (Abolfathi et al., 2004).

F. Lechin et al. / NeuroToxicology 27 (2006) 184–191 185

Littlework has been done to investigate the effects of centrally

acting drugs upon circulating neurotransmitters. Considering our

long experience measuring those parameters we feel that the

assessment of the effects of sibutramine on the latter will add

more information dealing with the neuropharmacological

mechanisms triggered by this drug. Such peripheral drug-

induced effectsmight explainmanyphysiological,metabolic and

clinical features occurring throughout neuropharmacological

treatments. This research, moreover, would help the under-

standing of complex central–peripheral interactions.

Neuropharmacological agents act on one or more CNS

circuits and all changes triggered at those levels provoke

neuroautonomic, neuroendocrine and behavioral effects

according to the subject’s pre-existing physiologic or patho-

physiologic state. Thus, full information about CNS circuitry

and CNS–peripheral interaction is basic to interpreting the

beneficial or adverse effects of all neuropharmacological drugs.

In view of the above, we proposed investigating the effects of

sibutramine, a potentially harmful drug widely used around the

world to treat obesity, and some syndromes linked to it.

2. Subjects and methods

We measured levels of plasma noradrenaline (NA), adrena-

line (Ad), dopamine (DA), free serotonin (f-5HT), tryptophane

(TRP), and platelet–serotonin (p-5HT) before (�30 and 0 min),

and after (60, 90, 120, 180, 240, 300, and 360 min) the oral

administration of 15-mg of sibutramine in 20 healthy

volunteers. We performed similar measurements 1 week earlier

in the same volunteers after administration of placebo. The

group of volunteers comprised 11 men and 9 women whose

ages ranged from 21 to 32 years (mean � S.E. = 25.8 � 2.3).

Informed consent was obtained in writing from all volunteers,

and the procedure was approved by the Ethical Committee of

FUNDAIME. All volunteers were within 10% of ideal body

weight, none had any physical or psychiatric illness. Exclusion

criteria included pregnancy, lactation, smoking, and alcohol

abuse. Volunteers were recumbent during all procedures. A

heparinized venous catheter was inserted into a forearm vein at

least 30 min before the test. We used cold, plastic syringes to

collect blood samples at the times specified above. Sibutramine

15 mg was administered orally after the second blood sample

(0 min) was obtained. Blood samples were obtained for

measuring plasma neurotransmitters and platelet aggregation.

Blood for measuring plasma neurotransmitters was transferred

to plastic tubes, each containing 1 ml of an anti-oxidant

solution (20 mg of EDTA plus 10 mg of sodiummetabisulphite/

ml). The tubes were carefully inverted several times and placed

on ice until centrifugation. To obtain platelet-rich plasma

(PRP), we centrifuged the tubes at 600 rpm at 4 8C for 15 min.

We stored 2 ml of PRP at �70 8C until needed for

determination of p-5HT levels. The remaining blood was

centrifuged again at 7000 rpm. We stored two aliquots of the

supernatant, which was platelet-poor plasma (PPP), at �70 8Cuntil needed for assays of catecholamine and f-5HT. Blood

samples for platelet aggregation were processed immediately.

A physician in constant attendance monitored heart rate and

blood pressure, and noted any symptoms reported by subjects.

2.1. Analytical assays

2.1.1. Neurochemistry

Plasma catecholamine and serotonin samples were mea-

sured in duplicate, and all determinations were made at the

same time. We used reverse phase, ion pair high-pressure liquid

chromatography with electrochemical detection (Davies and

Molyneux, 1982; Eisenhofer et al., 1986; Picard et al., 1985).

2.1.2. Reagents and standards

Noradrenaline, adrenaline, dopamine, serotonin creatinine

sulfate, dihydroxybenzylamine, 5-hydroxy-tryptophane, sodium

octyl sulfate, dibutylamine KH2PO4, citric acid, sodium acetate,

andEDTAwereobtained fromSigma-AldrichCo. (St.Louis,MO,

USA). Acid-washed aluminum oxide and microfilters were

purchased from Bioanalytical Systems Inc. (West Lafayette, IN,

USA).Acetonitrileand2-propanolwereobtainedfromRiedel-de-

Haen AG (Frankfurt, Germany). Glass-distilled water was

deionized and filtered through a Milli-O reagent grade water

system (Millipore, Bedford, MA, USA). Solutions and solvent

were filtered through a 0.2 mmMillipore filter and were vacuum

deaereated. Standard solutions (1 mmol/L) were prepared in

0.1 mol/Lperchloric acidanddiluted to thedesired concentration.

2.1.3. Equipment

Liquid chromatography was performed using Waters 515

pumps (Waters Co., Milford, MA, USA) equipped with 7125

Rheodyne valve injector fitted with a 50 ml sample loop for

detection of catecholamines, and 100 ml sample loop for p- and

f-5HT detection (Rheodyne, Berodine, Berkeley, CA, USA).

For catecholamine assays, a 15 cm � 4 mm i.d. DiscoveryTM

analytical column packed with C18 3 mm particles was used,

fitted with a precolumn filter 0.2 mm (Sigma-Aldrich Co., St.

Louis, MO, USA).

The detection system was a Waters 460 Electrochemical

Detector (Waters Co., Milford, MA, USA). A potential of

0.70 V was applied to the working electrode (glassy carbon)

versus the Ag–AgCl reference electrode. The chromatograms

were registered and quantified using Millennium software

(Waters Assoc., Milford, MA. USA).

2.1.4. Catecholamine assays

These were performed by extraction onto 20 mg of acid-

washed alumina followed by their elution with 200 ml of

0.2 mol/l HClO4 using BAS (Bioanalytical Systems) micro-

filters. The instrument was calibrated with standard plasma.

After incubation with acid-washed aluminum oxide, a plasma

pool free of catecholamines was obtained. This was processed

similarly to plasma samples, but 20 ml of standard solution

containing noradrenaline, adrenaline and dopamine (50 ng/ml

each) was added to l ml of the plasma pool to obtain the

standard plasma. Both standard plasma and sample plasma

were supplemented with 20 ml of internal standard solution

(dihydroxybenzylamine 100 ng/ml). The mobile phase was

F. Lechin et al. / NeuroToxicology 27 (2006) 184–191186

Fig. 1. Noradrenaline (NA), adrenaline (Ad), NA/Ad ratio and dopamine (DA)

in plasma before and after sibutramine administration. Values are mean � S.E.

composed of KH2PO4 50 mmol/L, EDTA 25.16 nmol/l, sodium

octyl sulfate 2.37 mmol/l, di-N-butylamine 100 ml/l, and

acetonitrile 2.5% (v/v) with pH adjusted to 5.2. Catecholamine

determinations were performed after injection of 50 ml of

processed plasma. Correction for dilution was performed.

Concentrations are expressed in pg/ml. The sensitivity of this

method for noradrenaline was 3.2 pg/ml, for adrenaline was 4.2

and for dopamine it was 2.5 pg/ml. The intra-assay coefficients

of variation were 2.3, 3.6, and 2.3% for noradrenaline,

adrenaline, and dopamine, respectively. The inter-assay

coefficients of variation were 2.6, 3.9, and 3.8%, respectively.

2.1.5. Serotonin assay

After sonication of PRP to disrupt any intact platelets

(Ultrasonic Liquid Processor, model 385, Heat Systems

Ultrasonic Inc., Farmingdale, NY, USA), both PRP and PPP

were processed in the same way: 200 m1 of 3.4 M perchloric

acid as deproteinizing agent and 10 ml of 5-OH-tryptophane

solution (80 mg/ml) as internal standard, were added to 1 ml of

plasma, vortexed and centrifuged at 10,000 rpm for 15 min at

4 8C. The clear supernatant was filtered through a 0.22 mm

membrane (Millipore) and injected in the HPLC. Calibration

runs were generated by spiking plasma blank containing 50 ml

of 5HT solution (10 mg/ml) and 10 ml of 5-OH-tryptophane

solution (80 mg/ml). This standard plasma was processed in the

same manner as samples. PRP serotonin (p-5HT) and PPP

serotonin (p-5HT) levels were determined after injection of

100 ml of the deproteinized sample onto a 30 cm � 4.0 mm i.d.

DiscoveryTM column filled with C18 5 mm. The mobile phase

was composed of citric acid 20 mmol, sodium acetate 50 mmol,

sodium octyl sulfate 6.45 nmol, dibutylamine 100 ml/l,

propanol 3.5% (v/v). PH was adjusted to 4.9, flow rate

0.70 m1/min. The sensitivity of this method for plasma

serotonin was 0.18 ng/ml; intra-assay coefficients of variation

were 2.8% for platelet-rich plasma serotonin and 3.1% for

platelet-poor plasma serotonin, respectively. Inter-assay coeffi-

cients of variation were 3.5 and 5.2%, respectively. Correction

factor for dilution was used. Concentrations are expressed in

ng/ml. Platelet serotonin value = PRP serotonin value (total

circulating serotonin) minus PPP serotonin value (f-5HT).

2.1.6. Platelet aggregation

Blood was collected with citrate–phosphate dextrose (1:9 v/

v) as the anticoagulant. Blood was subsequently centrifuged at

120 � g for 10 min to prepare PRP. Aggregation studies were

carried out according to Born’s method (1962), and aggregation

was induced by ADP and collagen at final concentrations of

4 mmol/l and 4 mg/ml, respectively. Maximal aggregation,

expressed as the percentage of maximal light transmission, was

measured.

2.2. Statistical analyses

Results are expressed as mean � S.E. Multivariate analyses

of variance with repeated measurements, paired t-test, and

correlation coefficients (exploratory factor analysis) were

employed in interpreting the data yielded by this investigation.

Differences were considered significant at p � 0.02. Dbase

StatsTM by Ashton Tate and SE by Abacus were used for

statistical analyses and Excel for graphics.

3. Results

Noradrenaline was significantly raised at all periods

following sibutramine administration. Maximal increases in

plasma noradrenaline occurred at 360 min period. Dopamine

plasma levels increased significantly. However, dopamine

increase tended to lessen at the 360 min period. Adrenaline

showed significant decreases at 360 and 240 min periods. The

NA/Ad ratio showed greatly significant and progressive

increases as of the first 60 min period (see Fig. 1). We noted

significant positive correlations between noradrenaline and

dopamine values between the 60 and 240 min periods.

However, no significant correlation was found at the

360 min period.

Although neither free serotonin in the plasma nor platelet

serotonin showed significant change throughout the 360 min of

the experimental trial, the f-5HT/p-5HT ratio did show

significant and progressive reduction from the 120 min period

until the end of the trial (see Fig. 2).

Plasma levels of tryptophane did not show significant

change throughout the experimental study (see Fig. 2).

3.1. Correlations

Significant positive correlations were found between NA

and NA/Ad ratio (r = 0.83; p < 0.001) and NA and DA

(r = 0.61; p < 0.01). The former tended to rise from the first to

F. Lechin et al. / NeuroToxicology 27 (2006) 184–191 187

Fig. 2. Plasma serotonin (f-5HT), platelet serotonin (p-5HT), plasma trypto-

phane, and f-5HT/p-5HT ratio before and after sibutramine administration.

Values are mean � S.E.

Fig. 3. Systolic blood pressure (SBP), diastolic blood pressure (DBP) and heart

rate before and after sibutramine administration. Values are mean � S.E.

the last (post sibutramine) period. The NA versus DA

correlation showed progressive fading and disappeared by

360 min period. These findings indicate that both NA and DA

arose from the same source (sympathetic nerves) rather than

adrenal glands. The progressive fading of the NA versus DA

positive correlation indicates that although both neurotrans-

mitters were released from the same terminals, predominance

of NA occurred during last period.

Noradrenaline versus DBP correlation values at post drug

periods were: 0.46*, 0.58**, 0.69***, 0.77***, and 0.81***

( p < 0.01; p < 0.005; p < 0.003, and p < 0.001, respectively.

Dopamine versus DBP positive correlations were also

significant at all but the fifth period. Values were: 0.42*,

0.47*, 0.53**, and 0.48*, respectively.

Significant and progressive negative correlations were found

between the NA/Ad ratio and the f-5HT/p-5HT ratio from the

120 min period until the trial’s end. No correlation was found

between f-5HT, p-5HT, and tryptophane values.

Close negative correlations were found between f-5HT/p-

5HT ratio and NA/Ad ratio: r: �0.49*, �0.65**, �0.72**,

�0.81***, and �0.84*** ( p < 0.05, p < 0.005, p < 0.005,

p < 0.05, and p < 0.005).

Neither heart rate nor systolic blood pressure showed

significant change throughout the experimental trial. Diastolic

blood pressure showed slight but significant increase from the

60 min period until 360 min period (see Fig. 3). Significant

positive correlations were found between NA/Ad and DBP

values, at those periods.

Significant negative correlations were found between DA

and DBP, at the two last periods 300 and 360 min.

No significant correlations were found between f-5HTand p-

5HT values. However, significant negative correlations were

found between f-5HT/p-5HT ratio and NA/Ad ratio at the 120,

180, 240, and 360 min periods.

Neither oral administration of sibutramine nor placebo

affected platelet aggregation in our healthy volunteers.

4. Discussion

In the present study we demonstrate that oral sibutramine

(15 mg) significantly increases noradrenaline (NA) levels in the

plasma. This effect is maintained throughout the duration of the

experimental protocol (6 h). Conversely, adrenaline (Ad)

showed no increase, rather a significant and persistent decrease

throughout the test. The above findings converge in a sharp

enhancement of the NA/Ad ratio. Considering that adrenal

glands secrete some 80% of Ad + 20% of NA + DA, our results

suggest that neural but not adrenal sympathetic activity is

stimulated by the drug.

The catecholamines and blood pressure changes triggered

by sibutramine fit well with the known pharmamacokinetic of

the drug (when administered during fasting state) (Abolfathi

et al., 2004). However, these effects should be different when

the drug was administered before eating. With respect to this,

we have previously demonstrated that both NA and p-5-HT

showed significant rises after oral glucose ingestion (Lechin

et al., 1992, 1993). In this case the NA increase is secondary to

the rise of insulin which crosses the blood brain barrier and

excites the LC-NA neurons. The rise of p-5-HT is secondary to

F. Lechin et al. / NeuroToxicology 27 (2006) 184–191188

the excitation of the enterocromafın cells triggered by glucose

plus the post-prandial parasympathetic drive. This latter

mechanism is absent after the administration of sibutramine.

Small but significant rises of DA were also found after the

drug’s administration. This finding is consistent with the well

known fact that both catecholamines arise from the same source

(sympathetic nerves). The fact that the NA versus DA

significant positive correlation registered in this study

diminished with time is consistent with the progressive

predominance of NA over DA release. It fits well with the

finding that NA/DA ratio tended to rise throughout the 6 h of the

trial. In this regard, it is well known that the DA pool which

exists at sympathetic terminals is released during neural

sympathetic activation (Soares Da Silva, 1986). The DA

released from those terminals acts as a modulatory agent able to

bridle overflowing NA release, by acting at DA2 inhibitory

auto-receptors existing at this level (Artalejo et al., 1985;

Mannelli et al., 1988; Missale et al., 1988; Soares Da Silva,

1986, 1987). Evidently, enhanced release of DA triggered by

the drug was not enough to impede an overwhelming release of

NA, responsible for the diastolic blood pressure (DBP) rise that

we registered in all cases. This phenomenon is the opposite of

that observed in the orthostatic hypotension syndrome when

excessive DA spillover impedes NA release from sympathetic

terminals (Robertson et al., 1979; Van Loon et al., 1979;

Wilffert et al., 1984; Ziegler et al., 1979). The above

observations are reinforced by our findings concerning blood

pressure versus NA, DA, and Ad correlations. Whereas DBP,

but not SBP, showed significant positive correlation versus NA

and DA, no significant correlation was observed between blood

pressure and Ad values.

Birkenfeld et al. (2005) found that resting blood pressure

tends to increase with sibutramine. These findings are similar to

those registered in our study. However, other contradictory

findings in the Birkenfeld et al. (2002, 2005) studies should be

discussed on the basis that peripheral sympathetic activity

depends on two separate branches: neural sympathetic

(sympathetic nerves = 90% NA and 10% DA) and adrenal

sympathetic (adrenal glands = 80% adrenaline + 10% noradre-

naline + 10% dopamine, approximately). These CNS sympa-

thetic drives may function in association or dissociation. Our

experimental design demonstrated that sibutramine triggers

neural but not adrenal sympathetic excitation. For this reason

diastolic but not systolic blood pressure showed significant

increase. These findings are consistent with those shown in our

study demonstrating that sibutramine did not raise either

systolic blood pressure or heart rate, both parameters depending

on beta-adrenergic (adrenal sympathetic) but not alpha-

adrenergic (neural sympathetic) mechanisms. The diastolic

blood pressure rise triggered by the drug showed attenuation

when ingested over several days (Birkenfeld et al., 2002), an

effect which should be attributed to the fact that sibutramine is

an inhibitor of both NA and 5-HT uptake. Obviously, the latter

effect is slower to be triggered than the former. Here, it should

be taken into account that dorsal raphe serotonergic neurons

send inhibitory axons to CNS-NA neurons (Szabo and Blier,

2001). These mechanisms explain why sibutramine-induced

anorectic effects, depending on serotonergic activity, increased

progressively.

At CNS level, the hyper-neural sympathetic activity

provoked by sibutramine in our experimental study fits well

with the postulation that this drug triggers hyperactivity of the

CNS-NA circuitry responsible for neural sympathetic but not

adrenal sympathetic activation. These findings are in accor-

dance with the fact that sibutramine is a central NA-uptake

enhancer drug. With respect to the above, it is well documented

that the two branches of peripheral sympathetic activity can

function in association or dissociation (Lechin et al., 2002b,

2004c; Young et al., 1984). Exhaustive experimental evidence

demonstrates that whereas adrenal sympathetic activity is

directly ruled by the medullary Ad-C1 nuclei, neural

sympathetic release depends on the noradrenergic pontine

nuclei (A5, A6, A7). Whereas the two C1-Ad nuclei send axons

to sympathetic pre-ganglionic neurons located at the cervical

and thoracic intermedio-lateral spinal column, the pontine

noradrenergic nuclei send axons to pre-ganglionic neurons of

the lumbar segment. Axons from the former group innervate the

adrenal glands whereas those of the second group reach the

lumbar sympathetic ganglia (Ader et al., 1979; Commissiong

et al., 1978; Dampney et al., 2003; Folkow and von Euler, 1954;

Gurtu et al., 1984; Hancock and Fougerousse, 1976; Kobayashi

et al., 1975; Nygren and Olson, 1977; Satoh et al., 1977; Smith

and Barrow, 1990; Pearson, 1978). Tracing with retrovirus has

ratified neurophysiological and neuropharmacological research

in this area (Blessing et al., 1981; Ding et al., 1993; Koulu et al.,

1986; Li and Guyenet, 1995; Li et al., 1992; Wessenlingh et al.,

1989). On the other hand, our research work dealing with the

assessment of circulating neurotransmitters before and after

various types of stress tests like orthostasis, exercise (Lechin

et al., 1994, 1995a,b, 1996b), oral glucose tolerance test

(Lechin et al., 1991, 1992, 1993), neuropharmacological drugs

(Lechin et al., 1997, 1985a,b), and others carried out during the

last 25 years, is in line with the clear demonstration that neural

and adrenal sympathetic activities depend on two distinct CNS

circuitries. One of the most important findings stemming from

the above is that diastolic blood pressure is closely and

maximally dependent on the CNS–pontine NA nuclei (A5, A6,

A7) activities, as demonstrated in the experimental model of

essential hypertension (Lewis rats) (Koulu et al., 1986). These

mammals are characterized by a hyperactive pontine NA

nuclei + DBP rise. In addition, they respond to all types of

emotional stimuli with enhancement of the firing of CNS-NA

neurons + diastolic blood pressure rise + increased NA levels at

both cerebro-spinal fluid and blood levels. According to the

foregoing, results emanating from the present investigation

strongly suggest that acute administration of sibutramine

provokes CNS-NA activation, responsible for the peripheral

neural sympathetic release without participation of adrenal

gland secretion. Our findings fit well with the fact that

sibutramine acts as an NA-uptake inhibitor.

The lowered f-5HT/p-5HT ratio provoked by sibutramine in

our experimental trial merits detailed discussion. Most

circulating serotonin is stored in platelets (p-5HT) = 97–

99%. Free serotonin in the plasma (f-5HT) accounts for the

F. Lechin et al. / NeuroToxicology 27 (2006) 184–191 189

remaining 1–3%. The f-5HT/p-5HT ratio increases during

platelet aggregation or platelet activation (Born, 1962). Other

factors able to increase f-5HT include the rise of plasma levels

of both acetylcholine (ACh) (Rausch et al., 1985) and dopamine

(DA) (De Keyser et al., 1988; Ricci et al., 2001). Both

neurotransmitters compete for platelet uptake with serotonin.

Thus, any rise of ACh and DA tends to increase f-5HT. The fact

that we found a reduction of the f-5HT/p-5HT ratio, in our

experimental subjects, despite noting moderate increases of DA

in the plasma, obliges us to postulate that ACh which also

interferes with platelet uptake, would be significantly lowered

after the drug’s administration. Although we cannot directly

measure ACh levels in peripheral blood, we previously

demonstrated a close positive correlation between f-5HT/p-

5HT ratio and peripheral parasympathetic activity in depressed

subjects (Lechin et al., 1995a,b, 1996a) known to present a

hyperparasympathetic profile. In addition, we showed that the

f-5HT/p-5HT ratio correlated positively with parasympathetic

activity occurring during sleep and wake periods (Lechin, 2000;

Lechin and van der Dijs, 2005; Lechin et al., 1992, 1994,

2004a,b) which was reverted by atropine (Lechin, 2000; Lechin

et al., 1994, 1996c). Thus, that ratio seems to be an adequate

index of ACh circulating levels in normal subjects, in absence

of platelet aggregation and other interfering factors. Accord-

ingly we propose that the very low f-5HT/p-5HT ratio observed

in our experimental study reflects a low parasympathetic

activity secondary to the neural sympathetic predominance

triggered by the drug. This postulation is supported by the fact

that the NA pontine nuclei send direct inhibitory axons to the

dorsal motor vagal nuclei (Lechin et al., 2002a). This

postulation is reinforced by the close negative correlations

we found in the present study between f-5HT/p-5HT ratio and

NA/Ad ratio. The low parasympathetic activity is also

consistent with the previously mentioned cardiovascular,

gastrointestinal, psychiatric, and sleep disturbances triggered

by the drug after prolonged administration (Birkenfeld et al.,

2002, 2005; de Simone et al., 2005; Jordan et al., 2005).

Considering that both p- and f-5-HT rises registered after an

oral glucose load (Lechin et al., 1992, 1993) reflects

parasympathetic activity, it is logical to assume that the

lowering of these parameters registered after sibutramine

administration should be associated with hypersympathetic

activity. In addition, it has been shown that the bioavailability of

sibutramine was significantly increased with the administration

of food (Abolfathi et al., 2004).

Sibutramine increases insulin sensitivity and improves

insulin resistance (Wu et al., 2003). With respect to this, it

is a well known fact that insulin crosses the blood brain barrier

and excites CNS-NA neurons. These CNS-NA neurons send

excitatory drive to neural sympathetic nerves which release NA

and DA. In addition, it has been demonstrated that insulin acts

as an NA-uptake inhibitor at the CNS-NA neurons (Boyd et al.,

1986). Thus, this drug behaves as a potentiating factor of the

CNS-induced insulin effect.

The fact that sibutramine decreased f-5HT/p-5HT ratio,

despite the fact that the drug is a serotonin uptake inhibitor (at

both platelets and serotonergic axons), reinforces the postula-

tion that this acute effect is secondary to the reduction of

circulating acetylcholine levels triggered by the enhancement

of neural sympathetic activity. These findings agree with the

postulation of the f-5HT/p-5HT ratio as an index of

parasympathetic activity in absence of platelet aggregation.

We investigated the effects of sibutramine throughout a 6-h

period. We did not prolonged this study because in hour

experience, prolonged supine-resting state tends to induce sleep

and might change the circulating neurotransmitters profile thus,

we would introduce an additional factor able to trigger both

central and peripheral neurochemical changes. However, we

are now investigating the neurotransmitters profile in subjects

who are consuming the drug during prolonged periods.

Summarizing all the above, it is logical to assume that

sibutramine reduces food intake by both acute and chronic

effects. The former would depend on the anti-parasympathetic

mechanisms triggered by the drug, whereas the enhancement of

central serotonergic activity would be responsible for the

chronic anorexigenic effects.

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